Dipeptidyl peptidase iv inhibitors for the treatment of schizophrenia and depression

ABSTRACT

A method for therapeutically treating an animal, including a human, for psychosomatic, depressive and neuropsychiatric diseases, such as anxiety, depression, insomnia, schizophrenia, epilepsy, spasm and chronic pain. Administration of a suitable DP IV inhibitor causes the reduction of activity in the enzyme dipeptidyl peptidase (DP IV or CD 26) or of DP IV-like enzyme activity in the brain of mammals and leads as a causal consequence to a reduced degradation of the neuropeptide Y (NPY) and similar substrates by DP IV and DP IV-like enzymes. Such treatment will result in a reduction or delay in the decrease of the concentration of functionally active neuronal NPY (1-36). As a consequence of the resulting enhanced stability of the endogenous NPY (1-36) caused by the inhibition of DP IV activity, NPY activity is prolonged thereby resulting among other things in functionally active NPY Y1 receptor activity thereby facilitating antidepressive, anxiolytic, analgesic, antihypertension and other neurological effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/397,281,filed Apr. 4, 2006, which is a continuation of application Ser. No.10/014,291, filed Oct. 26, 2001 and issued as U.S. Pat. No. 7,132,104 onNov. 7, 2006. The present application and all of the foregoingapplications claim the benefit of Provisional Application No. 60/224,036filed on Oct. 27, 2000. All of the foregoing applications areincorporated herein by reference in their entireties to the extentpermitted by law.

FIELD OF THE INVENTION

The present invention relates to the function of DPIV-like enzymeswithin the CNS and their biological effects on neuropeptide levels,neurotransmission and behavior. The present invention also relates tothe potentiation of endogenous neurological and neuropsychologicaleffects of brain neuropeptide Y (NPY) systems and other substrates ofDPIV by selective inhibition of DPIV-like enzymes. The invention relatesfurther to the treatment of hypertension, fever, sleep dysregulation,anorexia, anxiety related disorders including depression, seizuresincluding epilepsy, drug withdrawal and alcoholism, neurodegenerativedisorders including cognitive dysfunction and dementia, andneuropsychiatric disorders including schizophrenia, via a potentiationof NPY Y1 receptor mediated effects resulting from an inhibition ofDPIV-like activity within the CNS.

BACKGROUND OF THE INVENTION

CNS neuropeptide systems, peptide degradation and stress relateddiseases daptive responses initiate sequential steps of transmitterrelease in the CNS with corticotropin releasing hormone (CRH) being akey integrator (Dunn and Berridge, 1990; Koob and Heinrichs, 1999).Other neurotransmitters may modulate the course and outcome ofCRH-induced behavioral, endocrine and immunological alterations. It hasbeen demonstrated that endogenous neuropeptide Y (NPY) exertanti-CRH-like effects (Heilig et al., 1994; Thorsell et al., 1999;Britton et al., 2000). Recent clinical data implicate CRH in theetiology and pathophysiology of a variety of endocrine, psychiatric,neurodegenerative, and immunological disorders (Behan et al., 1995;Dieterich et al., 1997; Linthorst et al., 1997; Owens and Nemeroff,1991; Wilder, 1993). Apart from selective receptor blockade of CRHreceptors, an increase of endogenous anti-CRH-like acting NPY maytherefore be beneficial. The relative increase of an endogenous NPY-liketone may be pharmacologically achieved by either increased degradationof CRH or by inhibition of the degradation of NPY. NPY is a substratefor the enzyme DPIV. A modulation of CNS DPIV-like activity provides,therefore, a new treatment regime of neurological and neuropsychologicaldisorders.

DPIV and NPY

DPIV (CD26; EC 3.4.14.5) is an ectopeptidase with a triple functionalrole. DPIV is involved in truncation of Xaa-Pro dipeptides, circulatinghormones and chemokines (Mentlein et al., 1999; Pauly et al., 1999), inT cell dependent immune responses (Kahne et al., 1999; Korom et al.,1997) and in metastasis (Cheng et al., 1998; 2000). DPIV selectivelycleaves peptides after penultimate N-terminal proline and alanineresidues. Endogenous substrates for this enzyme include the incretins,such as glucose-dependent insulinotropic polypeptides, like GIP andGLP-1. In the presence of DP IV, these hormones are enzymically degradedto inactive forms. NPY is one of the best, if not the best, substratesof DPIV-like enzymically activity (Mentlein, 1999). So far, the functionof DPIV-like enzymatic activity within the CNS is not understood nor isthe modulation of CNS DPIV-like activity the objective of anypharmacological treatment regime.

Neuropeptide Y, peptide YY and pancreatic polypeptide share anevolutionary conserved proline-rich N-terminal sequence, a structuregenerally known to be inert to the attack of common proteinases, but apotential target for specialized proline-specific aminopeptidases.Mentlein et al. examined purified human DPIV, that liberated N-terminalTyr-Pro from both, neuropeptide Y and peptide YY, with very highspecific activities and K_(m) values in the micromolecular range, butalmost no Ala-Pro from pancreatic polypeptide. Other proline-specificaminopeptidases exhibited low (aminopeptidase P) or totally no activity(dipeptidyl peptidase II). When human serum was incubated withneuropeptide Y or peptide YY at micro- and nanomolar concentrations,Tyr-Pro was detected as a metabolite of both species. Formation ofTyr-Pro in serum was blocked in the presence of Lys-pyrrolidine anddiprotin A (Ile-Pro-Ile), specific competitive inhibitors of dipeptidylpeptidase IV. Incubation of neuropeptide Y or peptide YY withimmunocytochemically defined, cultivated endothelial cells from humanumbilical cord also yielded Tyr-Pro. Dipeptidyl peptidase IV could beimmunostained on most endothelial cells by a specific antibody. Theysuggest, that dipeptidyl peptidase IV might be involved in thedegradation of neuropeptide Y and peptide YY to N-terminal truncatedneuropeptide Y (3-36) and peptide YY (3-36). Since specific binding toY1, but not to Y2 subtype of neuropeptide Y/peptide YY receptorsrequires intact N- as well as C-termini of neuropeptide Y and peptideYY, removal of their amino-terminal dipeptides by dipeptidyl peptidaseIV inactivates them for binding to one receptor subtype (Mentlein et al.1993).

Discovery of NPY

Neuropeptide Y (NPY), a 36 amino acid peptide belonging to thepancreatic polypeptide family, was first isolated from porcine brain in1982 (Tatemoto and Mutt, 1982). NPY is present in all sympathetic nervesinnervating the cardiovascular system and is the most abundant peptidein the brain and the heart. Additionally, in rats, but not in humans,NPY is also found extraneuronally in platelets and endothelium(Zukovska-Grojec et al., 1993). Originally, NPY was known as a potentvasoconstrictor and a neuromodulator. Released by stress, exercise, andmyocardial ischemia, NPY has been implicated in coronary heart disease,congestive heart failure, and hypertension (Zukovska-Grojec et al,1998). More recently, because of the potent ability of NPY to stimulatefood intake, it is suspected to play a role in obesity and diabetes(Kalra et al., 1999). Latest findings indicate, that NPY is also amitogen for rat aortic vascular smooth muscle cells (Zukovska-Grojec etal., 1999).

NPY-related research has focussed on at least three main directions: (1)Co-transmission and sympathetic vasoconstriction, because of itsco-expression with noradrenaline; (2) neurotransmission and functionwithin the CNS, because of potent consummatory effects; and (3)evolution of NPY, since NPY is one of the most highly conservedbio-active peptides known (Colmers and Wahlestedt, 1993; Lundberg, 1996;Wahlestedt and Reis, 1993; Wettstein et al., 1996). NPY acts on at leastsix receptors (Y1-Y6), with varying peptide pharmacology and distinctdistribution in the CNS (Gehlert, 1998) (Tab. 1).

Distribution Of NPY NPY Receptor Subtypes and mRNA

The distribution of NPY itself, NPY receptor protein and their mRNAwithin the CNS of human and rat brains has recently been reviewed(Dumont Y, Jacques D, St-Pierre, J.-A., Tong, Y., Parker, R., Herzog H.and Qurion, R., 2000; in Handbook of Chemical Neuroanatomy, Vol. 16:Peptide Receptors, Part I; Quirion, R., Björklund, A. and Hökfeld, T.,editors). A brief survey is given in Tab. 1.

NPY-containing neurons are evident in the nasal mucosa of variousspecies including man, often associated with glandular acini and bloodvessels (Baraniuk et. Al., 1990; Grunditz et. al., 1994). Stimulation ofthe parasympathetic nerve supply to the nasal mucosa (vidian nerve) indogs increases blood flow in the region and causes mainly atropineresistance. Intravenous administration of NPY reduces vasodilitation dueto parasympathetic nerve stimulation, an effect that was not mimicked bythe NPY Y1-selective agonist [Leu31, Pro34]NPY, but was mimicked byadministration of the NPY Y2-receptor agonistN-acetyl[Leu28,Leu31JNPY(24-36) (Lacroix et al., 1994). This isconsistent with a prejunctional NPY Y2-like receptor-mediated inhibitionof transmitter release from parasympathetic nerve terminals.

NPY Receptor Function

Since the discovery of NPY in 1982, it became apparent that NPY isinvolved in the regulation of several behavioral and physiologicalfunctions (Colmers and Wahlestedt, 1993; Wettstein et al., 1996) (Tab.1). In the brain, NPY has been implicated in anxiety and depression,feeding and obesity, memory retention, neuronal excitability, endocrinefunction, and metabolism (Gehlert, 1998). NPY is unarguably the mostabundant neuropeptide discovered to date, with a wide distribution inthe CNS and the peripheral nervous system (PNS). NPY forms a family ofpeptides together with peptide YY (PYY) (approximately 70% homology) andpancreatic polypeptide (PP) (approximately 50% homology); both NPY andPYY are extremely bio-active, whereas PP is generally much less active(Gehlert, 1998; Wahlestedt and Reis, 1993) (Tab. 2).

Receptors for neuropeptide Y are also located on sensory nerve terminalsand their activation can modulate local neurogenic responses (Grundemaret al., 1990; 1993). Two receptor subtypes have been called neuropeptideY Y1 (postjunctional) and neuropeptide Y Y2 (prejunctional) on the basisof the different responses to a truncated analog of the related peptideYY- (13-36), when compared with neuropeptide Y in in vitro assay systems(Wahlestedt et al., 1986). Activation of neuronal prejunctional NPYreceptors generally inhibits nerve activity, reducing the release ofneurotransmitters in response to nerve impulses and in response to localfactors acting to release neurotransmitters (Wahlestedt et al., 1986).The prejunctional or neuropeptide Y Y2 receptor classification was basedon actions of peptide YY (13-36) but in many systems this molecule, aswell as neuropeptide Y-(13-36), does exhibit pressor activity (Rioux etal., 1986; Lundberg, et al., 1988; Potter et al., 1989). This has beeninterpreted by some to indicate that in some vascular beds there are twotypes of neuropeptide Y receptors (both neuropeptide Y Yj andneuropeptide Y2) on postjunctional membranes (Schwartz et al., 1989).However the lack of selectivity of these molecules may be due toretention of partial agonistic activity on Yj receptors, which permitsthem to evoke a reduced functional response. Previously, a 13-36 analogof neuropeptide Y, (Leu 17, Glu″, Ala 21, Ala 22, Glu 23, LeU28, LeU31)neuropeptide Y- (13-36) (ANA neuropeptide Y-(13-36)) which displayedprejunctional activity equivalent to the whole neuropeptide Y moleculein studies in vivo was described (Potter et al., 1989).

Apart from these historically well-defined neuropeptide Y receptors theexistence of a number of other subtypes (Y3, Y4, Y5 and Y6) has beensuggested on a pharmacological basis (Michel et al., 1998) and detailsof the cloning of receptors corresponding to Y1, Y2, Y4 and Y5 have beenpublished (Herzog et al., 1992; Gerald et al., 1995; Bard et al., 1995;Gerald et al., 1996) (Tab. 1). The distribution and physiologicalsignificance of these various receptor subtypes has yet to be defined.Although some controversy has existed about the selectivity of truncatedforms of neuropeptide Y for one or other receptor subtype (Potter etal., 1989), the emerging picture supports the initial classificationinto pre- and postjunctional receptor subtypes. Cell lines have beendeveloped which express specifically one neuropeptide Y receptor subtypeand the development of receptor-selective analogs of neuropeptide Y hasfocussed mainly on binding characteristics in these cell lines (Sheikhet al., 1989; Aakerlund et al., 1990; Fuhlendorff et al., 1990). Morerecently, a cDNA encoding the neuropeptide Y Y1 receptor has been clonedand cell lines expressing the cloned receptor have been analyzed forboth specific binding of neuropeptide Y analogs (Herzog et al., 1992)and functional responses elicited by specific analogs. From such bindingstudies, combined with subsequent studies in vivo, two analogs have beenclassified as acting specifically on the postjunctional neuropeptide YY1 receptor. These neuropeptide Y Y receptor selective analogs, (Pro 34)neuropeptide Y and (Leu″, Pro 34) neuropeptide Y, mimic the action ofneuropeptide Y in raising blood pressure, and also share similar bindingto cell lines expressing only neuropeptide Y Y receptors e.g. the humanneuroblastoma cell line SK-N-MC and fibroblast lines expressing thecloned neuropeptide Y Y, receptor (Herzog et al., 1992). Neitherexhibits the neuropeptide Y Y2 receptor action an inhibition of cardiacvagal action in vivo, a manifestation of inhibition of acetylcholinerelease (Potter et al., 1991; Potter and McCloskey, 1992).

TABLE 1 DISTRIBUTION AND FUNCTION OF NPY RECEPTOR SUBTYPES WITHIN THECNS Receptor- CNS Selective Antagonist subtype Expression FunctionSelective Agonist or Selectivity Y1 Cortex, etc. Anxiolysis, LHRH IntactN - Terminus: BIBP3226; BIBO 3304 Release [Leu31, Pro34]NPY Y2Hippocampus, Antiamnestic C-terminale End: T4[NPY(33-36)]4; HypothalamusPYY3-36; PYY13-36 BIIE0246 Y3 Ncl. Tractus Bradycardia, NPY>>PYY, PYY -Insensitivity Solitarius (NTS) Hypotension [Leu31, Pro34]NPY Y4 Dorsalvagal Emetic PP>>NPY, PYY PP - Preferring Complex (DVC) Y5 (a)Hypothalamus Feeding NPY, PYY, [Leu31, Pro34]NPY - [Leu31, Pro34]NPYsensitive, BIBP3226 - non-reversible Y5 (b) or Y6 Hypothalamus ?;species specific ? ? Tab. 1: NPY Receptor subtypes within the CNS; ? =unknown or not investigated

The development of the high affinity, non-peptide NPY antagonists,BIBP3226 and BIB03304, has facilitated the functional characterizationof NPY receptors, as this compound shows selectivity for Y1R, beingdevoid of activity on at least Y2R, Y3R and Y4R (Doods et al., 1996).Recently, a two Y2 receptor antagonist has been described. One is aTASP-molecule (Grouzmann et al., 1997), the other a non-peptideantagonist (Wieland et al., 1999) and other non-peptide receptorspecific compounds became available (Daniels et al., 1995). Thus,specific receptor blockade within the brain would allow the functionalcharacterization of behavioral and physiological effects mediated bycentral NPY receptors. In addition, mice lacking the Y1R were generatedand are available (Pedrazzini et al., 1998). Neurons showing NPY-likeimmunoreactivity and NPY receptor expression are abundant in the CNS(Tab. 1), and perhaps are most notably found in hypothalamic andso-called limbic structures, but are also co-localized with brain stemmonoaminergic neurons and cortical GABA-ergic neurons (Chronwall, 1985;Dumont et al., 1996). The latter may be of particular importance,because the GABA-benzodiazepine receptor complex is an importantnegative modulator of CRH secretion and of responsiveness to excitatorystimuli in rats and humans (Gear et al., 1997; Smith et al., 1992; Juddet al., 1995).

TABLE 2 RECEPTOR SUBTYPES UND PEPTIDE SELECTIVITY Receptor subtypePeptide Potency Y1-like Y1 NPY = PYY = Pro³⁴-NPY > PP > NPY₁₃₋₃₆ Y4PP >> NPY = PYY = LP-NPY > NPY₁₃₋₃₆ Y6 NPY = PYY = Pro³⁴-NPY >NPY₁₃₋₃₆ > PP Y2-like Y2 NPY = PYY = NPY₁₃₋₃₆ > Pro³⁴-NPY > PP Y5-likeY5 NPY = PYY = Pro³⁴-NPY > NPY₁₃₋₃₆ > PP Not cloned PP receptor PP >>PYY = NPY Y3 NPY = Pro³⁴-NPY = NPY₁₃₋₃₆ >> PYY PYY- PYY > NPY >>NPY₁₃₋₃₆ >> Pro³⁴-NPY preferring Tab. 2: Receptor subtypes and peptideselectivity according to Gehlert, 1998.

As has to be demonstrated, most of the central NPY effects are oppositeto those observed after CRH application, stress or those which are foundin anxiety related disorders. NPY almost completely resemble the effectsproduced by benzodiazepine application.

NPY And Autonomic Regulation

With respect to autonomic regulation, the results of Egawa et al. andothers on i.c.v. CRH- and i.c.v. NPY- (Egawa et al., 1990; 1991; vanDjik et al., 1994) mediated effects on sympathetic firing rate to brownadipose tissue (IBAT) demonstrate that CRH increases while NPY reducesthe sympathetic outflow. These effects support the anti-stress-likefunctional role of CNS NPY systems.

NPY And Immune Functions

The immune system is also affected by NPY. Here, similarly toCRH-mediated effects, the effects of NPY could be subdivided into directand indirect (centrally) mediated effects (von Hörsten et al., 1998a).I.c.v. applied NPY, and derived peptides, affect innate immune function,IL-6 levels, and leukocyte subsets, and these effects display dose,time, receptor, and compartment specificity (von Hörsten et al., 1998 a,b, c). Since NPY immunoreactivity increases in the brain afterperipheral induction of acute monoarthritis (Bileviciute et al., 1995),increased brain NPY levels may reflect partly an adaptive response tochanges induced by inflammation. Importantly, the long lastingimmunostimulatory action of i.c.v. NPY parallels the effects ofMethionine-Enkephalin (MET-ENK) (von Hörsten et al., 1998c). Thus, whilecentral CRH appears to be a key mediator of stress effects on the innateimmune system (Irwin, 1994), NPY may interact with CRH or evenantagonize its effects. The benzodiazepine-like action of NPY inconjunction with data demonstrating that benzodiazepines abrogateCRH-induced suppression of NK cell function (Irwin et al., 1993),further support the hypothesis, suggesting an anti-CRH-like,“stress-protective” action of NPY receptors activation.

NPY And Central Cardiovascular Regulation

The highest levels of NPY Y1, Y2, Y4 and Y5 receptors are found in thenucleus tractus solitarius (NTS), the area postrema and the dorsal vagalcomplex in the rat brain. These receptors are likely to be involved inthe CNS-mediated effects of NPY on various cardiovascular andrespiratory parameters (Dumont et al, 1992; McAuley et al., 1993). Forexample, direct injections of NPY into the NTS produce vasodepressoreffects and suppressed baroreceptor reflexes (Grundemar et al, 1992;Shih et al., 1992). These effects may be mediated via the Y2 or the Y3receptor based on the relative potency of NPY₁₃₋₃₆ (Narvaez et al, 1993)but no potency of PYY (Grundemar et al., 1991a, b). Thus, within theCNS, brainstem NPY systems may exert anti-hypertensive effects. Thesecentral effects of NPY appear to be opposite to the periphery. In theperipheral cardiovascular system, NPY raises blood pressure by an actionon postjunctional Y receptors and inhibits neurotransmitter release—bothacetylcholine and noradrenaline—by acting on prejunctional neuropeptideY receptors. When administered intravenously, NPY produces a potent andlong-lasting vasoconstriction that is not blocked by alpha or betaadrenergic antagonists (Wahlestedt et al., 1986).

NPY And Thermoregulation

Potent hypothermic effects of NPY have been described (Esteban et al.,1989; Jolicoeur et al., 1991; Currie and Coscina, 1995). Interestingly,Y1 receptor antisense-treated rats demonstrated increases in bodytemperature (Lopenz-Valpuesta et al, 1996), suggesting that the Y1receptor subtype could be responsible for the hypothermia induced byNPY. No further studies or pharmacological approaches explored thepossibility that NPY Y1 receptor activation might be useful in thetreatment of fever.

NPY, Circadian Rhythms and Sleep

The suprachiasmatic nucleus in conjunction with the geniculohypothalamic tract is of critical importance in diurnal rhythms (Albersand Ferris, 1984; Meijer and Reitveld, 1989). The effect of NPY oncircadian rhythms is believed to be mediated in the suprachiasmaticnucleus (Biello et a., 1994; Human and Albers, 1994) and the Y2 receptorsubtype has been implicated in the effect (Golombeck et al., 1996; Humanet al., 1996) by modulating glutamatergic neuronal activity (Biello etal., 1997). However, considering the effect of NPY on GABAergic neuronsin the suprachiasmatic nucleus (Chen and van den Poi, 1996; Biggs andProsser, 1999), it appears that other NPY receptor subtypes could alsoplay a role in modulation of circadian rhythms.

Disturbance of sleep is a common health problem and often associatedwith depression. In rats i.c.v. NPY treatment has been demonstrated toovercome CRH-induced and stress-induced shortening of sleep (Yamada etal., 1996). With regard to sleep regulation in humans, Ehlers et al.,(1997) found that “dysregulation” of sleep and arousal states indepression and anxiety may be consistent with an upset of the balancebetween hypothalamic neuropeptide systems for NPY and CRH. Antonijevicet al. (2000) reported that NPY promotes sleep and inhibits thehypothalamo-pituitary-adrenocortical (HP) axis in humans, pointing to apossible role of NPY agonists for the development of novel treatmentstrategies for affective disorders. Since in major depression increasedHPA-activity, sleeping disorder, anxiety and loss of appetite are maincharacteristics these findings further support a role of NPY inanxiety-disorders. Thus, a pharmacologically induced increased of NPYlevels might exert sleep-promoting effects. Yet, pharmacologicalapproaches to increase NPY levels are needed.

NPY And Nociception

Interestingly, NPY has also been reported to modulate nociception. Thereis evidence, that centrally (i.c.v.) applied NPY induces hyperalgesiceffects on hot plate latency in mice (Mellado et al., 1996) and rats(von Hörsten et al., 1998c). These results also parallel the findingthat benzodiazepines antagonize opioid and opiate analgesia via enhancedaction of GABA at the GABA-A receptors (Gear et al., 1997). At a spinallevel, in the dorsal root ganglia, NPY appears to exert analgesic-likeeffects and an increase of NPY Y2 receptor mRNA as well as NPY-likeimmunoreactivity has been reported after sciatic nerve lesions (Zhang etal., 1993). However, the physiological role of NPY in nociceptionremains to be established.

NPY And Feeding

On a behavioral level, most of the research has focussed on the potentorexigenic effects of NPY (Clark et al, 1984; Marsh et al., 1998; Kalraet al., 1999; O'Shea, et al., 1997; Stanley and Leibowitz, 1985). Theorexigenic effect of NPY parallels the known orexigenic “side” effect ofbenzodiazepine treatment, and is opposite to the anorexigenic effect ofCRH. CRH and NPY antagonize their feeding effects (Heinrichs et al.,1993; Menzaghi et al., 1993). I.c.v. CRH stopped weight gain ingenetically obese (fa/fa) NPY overexpressing rats (Bchini-Hooft et al.,1993), and it was shown that the hypothalamic NPY feeding system islargely dependent on circulating corticosterone (Stanley et al., 1989).Chronic i.c.v. infusion of NPY has been demonstrated to decreasehypothalamic content of CRH (Sainsbury et al., 1997). Possibly, theinduction of hunger by increased hypothalamic NPY content affects othermotivational systems. Structure-affinity and structure-activityrelationship studies of peptide analogs, combined with studies based onsite-directed mutagenesis and anti-receptor antibodies, have giveninsight into the individual characterization of each receptor subtyperelative to its interaction with the ligand, as well as to itsbiological function. A number of selective antagonists at theY1-receptor are available whose structures resemble that of theC-terminus of NPY. With respect to the behavioral regulation of feedingbehavior, some of these compounds, like BIBP3226, BIB03304 and GW 1229,have recently been used for in vivo investigations of the NPY-inducedincrease of food intake (Cabrele and Beck-Sickinger, 2000) and is wasfound that probably Y1 and Y5 receptors are involved in the mediation ofthese effects (Wieland et al., 1998).

-   -   NPY, Anxiety and Depression

Anxiolytic-like effects of NPY have been demonstrated using the elevatedplus maze test (Montgomery), the punished drinking test (Vogel), and thepunished responding test (Geller-Seifter), with potency and efficacymatching those of benzodiazepines (Griebel, 1999; Heilig et al., 1989;Wettstein et al., 1995). NPY acts anxiolytic-like on the response tonovelty (Heilig and Murison, 1987; von Hörsten et al., 1998b), andproduces anxiolytic-like effects on the elevated plus maze and otheranxiety related tests (Wahlstedt and Reis, 1993; Wahlestedt et al.,1993). Interestingly, Y1 receptor antisense-treated rats showed markedanxiety-related behaviors, without alterations of locomotor activity andfood intake (Wahlestedt et al., 1993). Additionally, in the Flintier ratstrain, a genetic model of depression, Y1 receptor mRNA expression wasdecreased in different cortical regions and the dentate gyms of thehippocampus, while Y2 receptor mRNA expression did not differ fromcontrols (Caberlotto et al., 1998). Olfactory bulbectomy in the rat hasbeen developed as a model of depression (Leonard and Tuite, 1981). Inthis model, most of the changes resemble those found in depressedpatients (Song et al., 1996). A 7-day i.c.v. administration of NPY inolfactory bulbectomized rats attenuated behavioral and neurotransmittersdeficits in this model (Song et al., 1996). All these data argue for arole of NPY in anxiety-related disorders. NPY Y1, Y2, and possibly Y5receptors, seem to be involved in the regulation of anxiety levels inrodents, with Y1-mediated effects being best characterized (Heilig etal., 1993; Kask et al., 1998b). Again, in comparison withbenzodiazepines, anxiolysis is one of the most important properties ofthese compounds, especially of those affecting central CRH systems (e.g.Alprazolam) (Arvat et al., 1998; Korbonits et al., 1995; Kravitz et al.,1993; Torpy et al., 1993). It can be concluded, therefore, thatendogenous NPY counteracts stress and anxiety (Heilig et al., 1994).Furthermore, these data suggest that the Y1 receptor subtype could beimplicated in anxiety- and depression-related behaviors. Additionally,Kask et al. (1996) reported that i.c.v. injection of the Y1 antagonist,BIBP3226, produced anxiogenic-like effects in the elevated plus-mazetest, without any locomotor deficit. This effect can be reproduced bythe administration of BIBP3226 in the dorsal periaqueductal gray matterbut not in the locus coeruleus o the paraventricular nucleus of thehypothalamus (Kask et al., 1998c). Moreover, BIBP3226 and GR231118administered into the dorsal periaqueductal gray matter decreased thetime spent in active social interaction in rats (Kask et al., 1998d).These data suggest that endogenous NPY, under stressful andnon-stressful conditions, relieve anxiety via the Y1 receptor.

The brain regions which are important for the anti-stress action of NPYinclude but may not be limited to the amygdala (Sajdyk et al., 1999,Thorsell et al., 1999), locus coeruleus (Kask et al., 1998c) and dorsalperiaqueductal gray (Kask et al., 1998a,b). Amygdala NPY is not releasedunder low stress conditions since blockade of NPY Y₁R with BIBP3226 orBIB03304 did not increase anxiety as measured in the elevated plus-mazeand social interaction tests (Kask et al., 1998b; Sajdyk, 1999).Constant NPY-ergic tone, however, seems to exist in the dorsalperiaqueductal gray matter, where the NPY Y₁R antagonist had anxiogeniclike effects in both experimental anxiety models (Kask et al., 1998a,b).Thus, in certain brain regions, there may be a tonic regulation ofanxiety via NPY systems.

Interestingly, the levels of NPY in the CSF of patients with majordepression were reduced as compared to non-depressed patients (Widerlovet al., 1986, 1988). Similarly, cortical tissues obtained form suicidevictims with a medical history of depression revealed lower levels ofNPY as compared to suicide victims with no reported depressive episodes(Widdowson et al., 1992). Higher levels of NPY in the CSF were found indepressed patients showing low symptoms of both psychological andsomatic anxieties, while anxious patients had lower levels (Heilig andWiderlov, 1995). Most recently, lower plasma levels of NPY were reportedin suicidal patients compared to healthy controls (Westrin et al.,1999). Taken together, these studies demonstrate that NPY is likelyinvolved in anxiety-related behaviors in humans. However, at present, nopharmacological approaches are available to gain advantage of thesebeneficial effects of elevated NPY levels in anxiety-related disorders.

NPY, Seizures and Epilepsy

Having the similarities between NPY mediated effects and benzodiapinesin mind, another important field for the treatment with benzodiazepinesis their anti-convulsive property. Surprisingly, NPY deficient mice,despite otherwise largely normal phenotypes, exhibit spontaneousseizures (Erickson et al., 1996a,b), while exogenously administered NPYreduces the incidence and severity of kainic acid-induced seizures(Woldbye et al., 1997). Elevated NPY levels have been observed followinglimbic seizures, suggesting that it may have a protective effect againstfurther seizure activity (Vezzani et al., 1996). Thus, another evolvingrole of NPY is found in neuronal excitability, and again, theparallelism with exogenous benzodiazepines is striking and the oppositeeffects to CRH-induced seizures (Ehlers et al., 1983) are apparent(Erickson et al., 1996; Vezzani et al., 1999).

In humans, temporal lob epilepsy is a neurological disorder in which thehippocampal formation is severely affected. In approximately two thirdsof the cases, the hippocampus is often the only structure that showspathological modifications (Amaral and Insausti, 1990). Considering thatNPY-containing neurons degenerate in the hippocampus of patients withtemporal lobe epilepsy (de Lanerolle et al., 1989) and that NPYregulates neuronal excitability in the rat hippocampus, the role of NPYand its receptors in humans certainly is critical. Thus, NPY and NPYreceptor provide an important pharmacological target for the developmentof new anti-epileptic-like acting drugs. However, no compoundsfulfilling pharmacological criteria for CNS targeting, peptide orreceptor specificity and bioavailability are available at present.

NPY, Learning, Aging, Neurodegeneration with Cognitive Dysfunction

NPY and PYY enhance memory retention (Flood et al., 1989). Thehippocampal Y2 receptor has been implicated in facilitating learning andmemory processes with increases in memory retention induced by NPY(Flood et at., 1987). Passive immunization with NPY antibodies injectedinto the hippocampus induced amnesia (Flood et al., 1989). Thehippocampal formation is associated with learning memory processes andis an area severely affected in Alzheimer's disease (Terry and Davies,1980). Several studies have reported significant decreases in NPY-likeimmunoreactivity in cortical, amygdaloid and hippocampal areas inAlzheimer's disease brains (Chan-Palay et al., 1986b). Moreover, NPYbinding sites are reduced in cortex and hippocampus of patientssuffering from Alzheimer's disease (Martel et al., 1990). These datasuggest that the degenerative process occurring in Alzheimer's diseasemay involve changes in NPY-related enervation. Interestingly, a majorloss in NPY-like immunoreactive neurons has been reported in aged ratsespecially in cortical areas (Cha et al., 1996, 1997; Huh et al, 1997;1998) and hypothalamic release of NPY is decreased in older rats(Hastings et al., 1998). The direct impact of NPY losses on cognitivebehaviors in Alzheimer's disease remains to be established. Similar, inother neurodegenerative disorders such as Huntington's disease selectivechanges of NPY changes have been reported (Ferrante et al., 1987; Bealet al., 1986). At present, no treatment approaches have focussed onincreasing NPY concentration in neurodegenerative disorders and/or otherdiseases states associated with cognitive dysfunction.

NPY, Opioid Withdrawal and Alcoholism

The expression of opioid withdrawal is thought to involve various brainregions (Koob et al., 1992). Early studies have suggested that exogenousapplication of NPY and related agonists could antagonize withdrawal bycorrecting deficits in NPY-like immunoreactivity at the levels of thehypothalamus (Pages et al., 1991). Recently, i.c.v. injections of NPYand related peptides have been shown to attenuated motor scoresalterations induced by naloxone-precipitated withdrawal from morphine inrats (Woldbey et al., 1998). It remains to be established if similardata could be obtained in humans. Very recently, experimental studieshave suggested that NPY, together with its receptors, may have directimplication in addiction to alcohol. NPY is involved in the Modulationof ethanol consumption and resistance. NPY-knockout mice were shown tohave high ethanol consumption and low sensitivity to ethanol (Thiele etal., 1998). In contrast, mice overexpressing NPY drank much less alcoholthan wild type and were also more sensitive to ethanol (Thiele et al.,1998). At present, no pharmacological approaches are available to gainadvantage of these likely beneficial effects of elevated NPY levels inwithdrawal and alcoholism.

NPY And Schizophrenia

Emerging evidence suggests that NPY might be involved in thepathophysiology of neuropsychiatric disorders (Wettstein et al., 1995).Region-specific decrease in NPY content has been described in patientswith schizophrenia (Frederikson et al., 1991; Widerlov et al., 1988).Overactivity of mesolimbic dopaminergic pathways is believed to be ofimportance in drug reinforcement and schizophrenia (Beninger, 1983).There is evidence that some effects of NPY might be mediated viaactivation of dopamine (DA) receptors. First, a number of behavioraleffects of NPY can be blocked by DA receptor antagonists (Moore et al.,1994; Josselyn and Beninger, 1993). Moreover, NPY has been shown tostimulate NMDA-stimulated DA release from rat striatum (Ault andWerling, 1997) and nucleus accumbens (Ault et al., 1998) providingdirect evidence that NPY potentiates dopaminergic neurotransmission inthese brain regions. Kask and Harro (2000) found that NPY Y1 receptorantagonism inhibits amphetamine-induced hyperactivity in rats andconcluded that the ability of NPY Y1 receptor selective antagonists tomodulate behavioral response to amphetamine an apomorhine suggests thatNPY Y1 receptors may be involved in mediation of psychosis andreinforcement.

Neurological and Psychophysiological Effects of CNS NPY SystemsPleiotropy

Thus, numerous studies have addressed the physiological functions of NPYand its congeners in the CNS (for reviews see: Kalra and Crowley, 1992;Dumont et al., 1992; Stanley, 1993; Wahlestedt and Reis, 1993; Grundemaret al, 1993; Gehlert, 1994, 1998; Colmers and Bleakman, 1994; Wettsteinet al, 1995; Heilig and Widerlowi, 1995; Munglani et al., 1996; Inui,1999; Bischoff and Michel, 1999; Vezzani et al., 1999) and demonstrateda broad range of effects. This pleiotropy, together with its high degreeof identity among mammalian species, suggests that NPY systems withinthe CNS are highly important pharmacological targets in variouspathophysiological states. The peptide is involved in severalneurological and psychophysiological processes, of which theanxiolytic-like, feeding, anti-convulsive and anti-addictive effectsappear to be most prominent. These actions involve a site specific and areceptor specific action of NPY within the CNS. No pharmacologicalapproaches exist, at present, to gain advantage of these variousphysiological functions.

Current Treatments of Anxiety Related Disorders Using Benzodiazepines

There are a number of new anxiolytic drugs that are fast acting and freefrom the unwanted side effects associated with the traditionalbenzodiazepines on the way toward the clinical development. Partialagonists at the benzodiazepine receptor, such as alpidem, abecarnil andbretazenil, have highly promising preclinical profiles and some usefulpreliminary results in clinical testing of anxiety disorder subjects.Neurosteroids are another interesting set of pharmacologic agents thattarget the benzodiazepine receptor, have a preclinical anxiolyticprofile and now need to be tested in clinical populations.

Neuropeptide receptor agonists and antagonists with anxiolyticproperties may represent one of the most striking new classes of thepotential anxiolytic drugs. As described above in detail, preclinicalstudies as well as clinical studies suggest that agonists of theneuropeptide Y receptors are provocative targets for anxiolytic agents(Kunovac & Stahl, 1995).

Current Problems in the Treatment of Anxiety Related Disorders UsingBenzodiazepines or NPY

The current methods for treatment of anxiety are accompanied by severalproblems:

The benzodiazepines that are commonly used as anxiolytic agents areunnatural compounds with a low or no selectivity. Beside theiranxiolytic activity, the benzodiazepines show sedative andanti-epileptic effects and are suspected to influence muscle relaxation.Unfortunately, they are associated with a number of unwanted sideeffects, namely tiredness, sleepiness, lack of concentration, reductionof attentiveness and reactivity. Chronic application of benzodiazepinescauses neurological disorders, like ataxia, dizziness, reflex loss,muscle and language disorders. A long-term treatment withbenzodiazepines is predicted to entail dependency and addiction.

The direct i.c.v. administration of neuropeptide Y for the long-termtreatment of anxiety in patients is not feasible.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide the beneficialneurological and psychophysiological effects that result from theinhibition of DPIV-like enzymatic activity within the CNS. In addition,it is an object of the present invention to overcome or reduce the abovestated problems with the prior art by providing a pharmacologicalapproach that mimics a reduced DPIV-like enzymatic activity within theCNS and results in a delayed degradation of NPY or any other DPIV-likesubstrate. Further provided is one mechanism of action that—via aninhibition of the brain DPIV-like enzymatic activity—results in themagnification of endogenous neurological or neuropsychological effectsmediated by NPY Y1 receptors, including but not limited to a reductionof anxiety. Also provided is a method of treating hypertension, fever,sleep dysregulation, anorexia, anxiety related disorders includingdepression, seizures including epilepsy, drug withdrawal and alcoholism,neurodegenerative disorders including cognitive dysfunction anddementia, and neuropsychiatric disorders including schizophreniadiagnosed in a subject, compromising administering to the subject a CNStargeted DPIV inhibitor via oral, parenteral or any other route ofadministration.

One aspect of the invention is directed to a method of treatingschizophrenia and/or depression. Such method comprises administering toa subject in need thereof, a therapeutically effective amount of acomposition comprising an inhibitor of dipeptidyl peptidase IV (DP IV),or a pharmaceutically acceptable salt thereof. In some embodiments, themethod of treatment is for schizophrenia. In some embodiments, themethod of treatment is for depression. In some embodiments, the methodof treatment is for schizophrenia and depression.

In some embodiments, the administration is oral. In some embodiments,administration is parenteral. In some configurations, parenteraladministration is intravenous. In some configurations, parenteraladministration is by injection. In some configurations, parenteraladministration is by infusion. In some configurations, parenteraladministration is by inhalation. In some embodiments, the administrationis by suppository.

In some embodiments, the inhibitor is N-(N′-substitutedglycyl)-2-cyanopyrrolidines; L-threo-isoleucyl thiazolidine;L-threo-isoleucyl pyrrolidine; L-allo-isoleucyl thiazolidine; orL-allo-isoleucyl pyrrolidine.

In some embodiments, the inhibitor is present in a physiologicallycompatible drug delivery vehicle.

In some embodiments, the method further comprises administeringneuropeptide Y to the patient.

In some embodiments, the composition comprises a prodrug form of the DPIV inhibitor.

In some embodiments, the neuropeptide Y and the inhibitor of dipeptidylpeptidase IV (DP IV) are administered simultaneously. In someembodiments, the composition further comprises neuropeptide Y. In someembodiments, the neuropeptide Y and the inhibitor of dipeptidylpeptidase IV (DP IV) are administered separately.

In some embodiments, the composition further comprises at least onepharmaceutically acceptable carrier or excipient.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 shows the DPIV enzymatic activity in serum of Fischer 344 (F344)rat substrains from Hannover (HAN), United States (USA), German (GER)and Japanese (JAP) breeders. The results are mean (±SEM) of 4-5age-matched animals per genotype. Analysis of variance revealed asignificant effect of “substrain” with F(3, 15): 50.4, p<0.0001.Asterisks indicate significant PLSD post hoc effects vs. “wild-type”F344USA and F344HAN substrains (“***”=p<0.0001).

FIG. 2 shows the time spent in active social interaction (SI) time inFischer 344 (F344) rat substrains from Japanese (JAP), United States(USA) and German (GER) breeders. F344JAP and F344GER rats are mutant forthe enzymatic function of dipeptidyl-peptidase IV (DPPIV) and lackendogenous DPPIV activity (see also FIG. 1). F344JAP rats provide a“protein knock-out” model because DPPIV is markedly reduced expressed oncell surface (Tsuji et al., 1992; Cheng et al., 1999). F344GER ratsexpress a mutant non-enzymically active DPPIV-like protein (Thompson etal., 1991). An increase of the Si time in the rat social interactiontest of anxiety is interpreted as an anxiolytic-like response. Theresults are mean (±SEM) of 12 age-matched animals per genotype. Analysisof variance revealed a significant effect of “substrain” with F(2, 32):8.8, p=0.0009. Asterisks indicate significant PLSD post hoc effects vs.“wild-type” F344USA rats (“**”=p<0.01; “***”=p<0.001).

FIG. 3 shows the change of body weight after stress on three consecutivedays. On three consecutive days, age matched animals from Japanese(JAP), United States (USA) and German (GER) breeders were individuallytransported to a novel room and remained there for 1 h. On the first daya novel cage containing sawdust was used and animals placed in astandard animal rack. On the second day procedure was the same exceptthat the cage was without sawdust. The stress procedure on the third wasthe same as on day 2 except that the cage was placed on the bottom ofthe novel room. Analysis of variance for repeated measures revealed asignificant effect of “substrain” with F(2, 30): 13.5, p=0.0004.Asterisks indicate significant PLSD post hoc effects one factor ANOVAssplit by day vs. “wild-type” F344USA (“*”=p<0.05; “**”=p<0.01).

FIG. 4 shows the effect of i.c.v. P32/98 treatment on the distancetraveled in four consecutive minutes of open field testing. Analysis ofvariance for repeated measures revealed no significant effect oftreatment on this parameter of activity (F(3, 78): 0.7, p=0.5; n.s.).

FIG. 5 shows the effect of i.c.v. P32/98 treatment on the time spentclose to the wall as a sum of four consecutive minutes of open fieldtesting. Analysis of variance revealed a significant effect of“treatment” with F(3, 26): 4.1, p=0.015. Asterisks indicate significantPLSD post hoc effects vs. “aCFS” controls (“*”=p<0.05).

FIG. 6 shows the effect of i.c.v. P32/98 treatment on the percentage oftime spent on the open arms of the elevated plus maze (EPM). Analysis ofvariance revealed a significant effect of “treatment” with F(3, 26):3.0, p=0.048. Asterisks indicate significant PLSD post hoc effects vs.“aCFS” controls (“*”=p<0.05).

FIG. 7 shows the effect of combined i.c.v. treatment using aCSF, P32/98and NPY in combinations at different dosages (P32/98: 5 pmol-500 nmol;NPY: 50 pmol-1.6 nmol). The time spent in active social interaction(SI-time) in the social interaction test of anxiety was measured. Anincrease of the SI-time is indicative for an anxiolytic like effect.After habituation to the testing procedure animals were repeatedlytested with randomly chosen treatment and always-new interactionpartners. Tests were separated with at least 4 days from each other. Foreach test, spanning four groups of 5-6 animals per condition, analysisof variance revealed the following significant effects of “treatment”(aCSF+aCSF; aCSF+NPY; P32/98+aCSF; P32/98+NPY) from left to right:P32/98 5 pmol+NPY 50 pmol: F(3; 18): 0.25, p=0.8, n.s.; P32/98 50pmol+NPY 100 pmol: F(3, 18): 22.4, p<0.0001; P32/98 500 pmol+NPY 200pmol: F(3, 20): 8.6, p=0.007; P32/98 50 nmol+NPY 0.8 nmol: F(3, 20):23.3, p<0.0001; and P32/98 500 nmol+NPY 1.6 nmol: F(3, 20): 11.2,p=0.0008. Asterisks indicate significant PLSD post hoc effects vs.“aCFS+aCSF’ controls and as indicated by bars between aCSF+NPY vs.P32/98+NPY (“*”=p<0.05).

FIG. 8 shows the effect of combined i.c.v. treatment using aCSF, P32/98and NPY in combinations at different dosages (P32/98: 5 pmol-500 nmol;NPY: 50 pmol-1.6 nmol). The amount of food eaten within 1 h wasmeasured. Animals were repeatedly tested with randomly chosentreatments. Tests were separated with at least 4 days from each other.For each test, spanning four groups of 5-6 animals per condition,analysis of variance revealed the following significant effects of“treatment” (aCSF+aCSF; aCSF+NPY; P32/98+aCSF; P32/98+NPY) from left toright: P32/98 5 pmol+NPY 50 pmol: F(3, 18): 7.0, p=0.0025;; P32/98 50pmol+NPY 100 pmol: F(3, 20): 4.5, p=0.016; P32/98 500 pmol+NPY 200 pmol:F(3, 20): 4.4, p=0.015; P32/98 50 nmol+NPY 0.8 nmol: F(3, 20): 6.6,p=0.0027; and P32/98 500 nmol+NPY 1.6 nmol: F(3, 20): 13.7, p<0.0001.Asterisks indicate significant PLSD post hoc effects vs. “aCFS+aCSF”controls and as indicated by bars between aCSF+NPY vs. P32/98+NPY(“*”=p<0.05).

FIG. 9 shows the effect of combined i.c.v. treatment using aCSF, P32/98and NPY in combinations at different dosages (P32/98: 5 pmol-500 nmol;NPY: 50 pmol-1.6 nmol). The amount of food eaten within 12 h wasmeasured. For each test, spanning four groups of 5-6 animals percondition, analysis of variance revealed the following significanteffects of “treatment” (aCSF+aCSF; aCSF+NPY; P32/98+aCSF; P32/98+NPY)from left to right: P32/98 5 pmol+NPY 50 pmol: F(3, 18): 0.5, p=0.7,n.s.; P32/98 50 pmol+NPY 100 pmol: F(3, 20): 0.17, p=0.9, n.s.; P32/98500 pmol+NPY 200 pmol: F(3, 20): 1.1, p=0.34, n.s.; P32/98 50 nmol+NPY0.8 nmol: F(3, 20): 1.2, p=0.3; and P32/98 500 nmol+NPY 1.6 nmol: F(3,20): 3.4, p=0.039. Asterisks indicate significant PLSD post hoc effectsvs. “aCFS+aCSF” controls and as indicated by bars between aCSF+NPY vs.P32/98+NPY (“*”=p<0.05).

FIG. 10 shows the effect of combined i.c.v. treatment using the Y1Rantagonist BIBP3226, P32/98 and NPY in combinations (BIBP3226: 100 nmol;P32/98: 50 nmol; NPY: 0.8 nmol). The time spent in active socialinteraction (SI-time) in the social interaction test of anxiety wasmeasured. An increase of the SI-time is indicative for an anxiolyticlike effect. After habituation to the testing procedure animals wererandomly assigned to i.c.v. treatment protocols and same treatmentinteraction partners. Tests spanned two consecutive with a total of 6-8animals per treatment condition. Analysis of variance revealed asignificant effect of “treatment” with F(7, 44): 33.6, p<0.0001 spanningthe following groups: (1) aCSF+aCSF+aCSF; (2) BIBP+aCSF+aCSF; (3)aCSF+P32/98+aCSF; (4) BIBP+P32/98+aCSF; (5) aCSF+aCSF+NPY; (6)BIBP+aCSF+NPY; (7) aCSF+P32/89+NPY; (8) BIBP+P32/98+NPY. The level ofsignificance in post hoc comparisons vs. controls (aCSF+aCSF+aCSF) isindicated by asterisks with “*”=p<0.05; “**”=p<0.01; “***”=p<0.001) andvs. corresponding antagonistic treatment (BIBP+n.n.) by “#” symbols with“#”=p<0.05; “##”=p<0.01; “###”=p<0.001). All data are presented asmeans±S.E.M.

FIG. 11 shows the effect of combined i.c.v. treatment on 1 h food-intakeusing the Y1 R antagonist BIBP3226, P32/98 and NPY in combinations(BIBP3226: 100 nmol; P32/98: 50 nmol; NPY: 0.8 nmol). After habituationto the testing procedure animals were randomly assigned to i.c.v.treatment protocols and same treatment interaction partners. Testsspanned two consecutive with a total of 6-8 animals per treatmentcondition. Analysis of variance revealed a significant effect of“treatment” with F(7, 44): 5.4, p<0.0002 spanning the following groups:(1) aCSF+aCSF+aCSF; (2) BIBP+aCSF+aCSF; (3) aCSF+P32/98+aCSF; (4)BIBP+P32/98+aCSF; (5) aCSF+aCSF+NPY; (6) BIBP+aCSF+NPY; (7)aCSF+P32/89+NPY; (8) BIBP+P32/98+NPY. The level of significance in posthoc comparisons vs. controls (aCSF+aCSF+aCSF) is indicated by asteriskswith “*”=p<0.05; “**”=p<0.0l; “***”=p<0.001) and vs. correspondingantagonistic treatment (BIBP+n.n.) by “#” symbols with “#”=p<0.05;“##”=p<0.01; “###”=p<0.001). All data are presented as means±S.E.M.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

In contrast to other proposed methods in the art, the present inventionprovides an orally available therapy with low molecular weightinhibitors of dipeptidyl peptidase IV. The instant invention representsa novel approach for the treatment of anxiety and other neurological orpsychological disorders. It is user friendly, commercially useful andsuitable for use in a therapeutic regime, especially concerning humandisease.

Examples for orally available low molecular weight dipeptidyl peptidaseIV inhibitors are agents such as, N-(N′-substitutedglycyl)-2-cyanopyrrolidines, L-threo-isoleucyl thiazolidine,L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine,L-allo-isoleucyl thiazolidine, and L-allo-isoleucyl pyrrolidine. Theyare described in U.S. Pat. No. 6,001, 155, WO 99/61431, WO 99/67278, WO99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO00/07617, WO 99/38501, and WO 99/46272, the teachings of which areherein incorporated by reference in their entirety. The goal of theseagents is to inhibit DPIV, and by doing so, to lower blood glucoselevels thereby effectively treating hyperglycemia and the attendantdisease associated with elevated levels of glucose in the blood.

DPIV is an enzyme that is an exopeptidase, which selectively cleavespeptides after penultimate N-terminal proline and alanine residues.Endogenous substrates for this enzyme include the incretins, such asglucose-dependent insulinotropic polypeptides, like GIP and GLP-1. Inthe presence of DPIV, these hormones are enzymically reduced to inactiveforms. The inactive form of GIP and GLP cannot induce insulin secretion,thus blood glucose levels are elevated, especially in the hyperglycemicstate. Elevated blood glucose levels have been associated with manydifferent pathologies, including diabetes mellitus (Type 1 and 2) andthe sequel accompanying diabetes mellitus.

It has also been discovered that DPIV plays a role in T-cell-mediatedimmune responses, for example, in organ transplantation. Inhibition ofDPIV has been demonstrated to prolong cardiac allografts. Additionally,the inhibition of DPIV has contributed to the suppression of rheumatoidarthritis. DPIV has also been attributed a role in HIV's penetrationinto T-cells (T-helper cells).

These various effects of dipeptidyl peptidase IV inhibitors imply theirimpact on normal healthy tissues and organs, when they are used for thetreatment of a pathologically altered tissue. The goal of the presentinvention is the development of highly selective brain targeteddipeptidyl peptidase IV inhibitors, which display a high bioavailabilityand an exactly predictable activity time in the target tissue.

Examples for target specific, orally available low molecular weightagents are prodrugs of stable and unstable dipeptidyl peptidase IVinhibitors which comprise general formula A-B-C, whereby A represents anamino acid, B represents the chemical bond between A and C or an aminoacid, and C represents an unstable or a stable inhibitor of dipeptidylpeptidase IV respectively. They are described in WO 99/67278, WO99/67279 the teachings of which are herein incorporated by reference intheir entirety.

The present invention relates to a novel method in which the reductionof activity in the enzyme dipeptidyl peptidase (DPIV or CD 26) or ofDPIV-like enzyme activity in brain of mammals induced by effectors ofthe enzyme leads as a causal consequence to a reduced degradation of theneuropeptide Y (NPY) by DPIV and DPIV-like enzymes. Such treatment willresult in a reduction or delay in the decrease of the concentration offunctional active NPY (1-36).

As a consequence of the resulting enhanced stability of the endogenousNPY (1-36) caused by the inhibition of DPIV activity, NPY activity isprolonged resulting in functionally active NPY Y1 receptor activityfacilitating—among others—anti-depressive, anxiolytic andanti-hypertensive effects (see above).

The method of the present invention for treating anxiety in an animal,including humans, in need thereof, comprises potentiating NPY's presenceby inhibiting DPIV, or related enzyme activities, using an inhibitor ofthe enzyme. Oral administration of a DPIV inhibitor may be preferable inmost circumstances. By inhibiting the DP IV enzyme activity, thehalf-life of the active form of NPY will be appreciably extended andmaintained under physiological conditions. The extended presence ofactive NPY will enhance the NPY Y1 receptor activity.

This invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically (or prophylactically) effectiveamount of the inhibitor (and/or a sugar pill to accompany administrationof a DPIV inhibitor), and a pharmaceutically acceptable carrier orexcipient. Suitable carriers include but are not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The carrier and composition are preferably produced under goodlaboratory practices conditions and most preferably are sterile. Theformulation is ideally selected to suit the mode of administration, inaccordance with conventional practice.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (for example, NaCl), alcohols, gumarabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch; magnesium stearate,talc, viscous paraffin, perfume oil, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, for example, lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds, but which improvestability, manufacturability and/or aesthetic appeal.

The compositions, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. In addition, thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. In addition,the composition can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodiumsaccharine, cellulose, magnesium carbonate etc.

Further, the compositions can be formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active compound. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water, salineor dextrose/water. Where the composition is administered by injection,an ampoule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

Finally, compositions of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree amino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acid, etc., and those derived from sodium,potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the invention's composition which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro and/or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgement of the practitioner andeach patient's circumstances.

It will be readily understood by the skilled artisan that numerousalterations may be made to the examples and instructions given hereinincluding the generation of different DP IV inhibitors and alternatetherapeutic compositions without departing from either the spirit orscope of the present invention.

The present invention will now be illustrated with reference to thefollowing examples focussing on the anxiolytic-like andstress-protective-like action of reduced DPIV-like activity in a geneticmodel of DPIV deficiency (example 1), the anxiolytic-like action ofpharmacologically induced DPIV inhibition within the CNS (example 2),the interaction and potentiation of NPY mediated anxiolytic-like effects(example 3), and the characterization of an anxiolytic-like mechanismcompromising DPIV-inhibition and resulting potentiation of NPY Y1receptor mediated effects (Example 4).

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that all examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES OF THE INVENTION

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1

Spontaneous mutations of the DPIV gene observed in substrains of Fischer(F344) rats provide a model for studying the role of DPIV in behavioralregulation and adaptation to stress. The mutations in F344 rats resultin a lack of DPIV-like enzymatic activity and are found in substrainsfrom Germany (GER) and Japan (JAP) (Thompson et al., 1991; Tsuji et al.,1992) while rats from US (USA) and Hannover (HAN) breeders showsignificant enzyme activity. In F344JAP rats, a G633R substitution inthe CD26 protein causes a greatly reduced expression of a mutantinactive enzyme (Tsuji et al., 1992; Cheng et al., 1999) while the otherDPIV negative F344GER substrain expresses a non-active mutant enzyme(Thompson et al., 1991). The F344JAP rat is therefore be considered asan “protein knock-out” model (Cheng et al., 1999) while the F344GERsubstrain may represent a “protein over-expression” model (Shingu,Helfritz, Meyer, Schmidt, Mentlein, von Hörsten, submitted). On thebasis of these findings, a direct comparison of mutant F344JAP andF344GER substrains with “wild-type” F344USA rats would allow thedifferentiation between the role of DPIV expression and activity onbehavioral regulation and other neurological and psychophysiologicalfunctions in vivo. In the present example we report that DPIV deficientF344 substrains are less anxious and less responsive to stress-inducedphysiological changes.

Animals. F344USA, F344JAP and F344GER substrains were obtained from thedifferent countries via Charles River Germany. F344Han rats, initiallyderived from the F344USA substrain, were obtained from a breeding colonyat the Central Animal Laboratory at Hannover Medical School. Allsubstrains were bred for one generation at the Central Animal Laboratoryat Hannover and maintained in a specific-pathogen-free facility at 25°C. under a 12 h light-12 h dark cycle (light on at 0700 h), with adlibitum access to food and water. For the experiments age-matched weeksold F1 offspring of all substrains was used. The District Government,Hannover, Germany, approved all research and animal care procedures.

Quantification of DPIV activity in tissue of F344 substrains. Plasma,lung and various other tissue samples were kept frozen at −80° C. untiluse. Tissue was homogenized and DPIV enzyme activity was detected byincubating the substrate, glycylproline p-nitroanilide (gly-Pro-pNA, 1mg/ml in PBS) (Bachem, Germany), and the color development was measuredat 405 nm.

Social interaction (SI) test. The SI test was carried out as firstdescribed by File (1980) and has initially been validated in thelaboratory (Kask, Nguyen, Pabst, von Hörsten, submitted). Two rats,matched for genotype and body weight, were removed from home cages andexposed to the testing environment. The arena was a squared open field(50×50×50 cm) made of aluminum, placed inside a sound isolation box(Coulbourn Instruments, Lehigh Valley, Pa.). For details of theapparatus see our previous study (von Hörsten et al., 1998c). The openfield was lit by a red photo light bulb (Philips PF712E; 1.3 Lux). Ratswere unfamiliar with the apparatus. Behavior was monitored using a videocamera placed above the field inside the testing/isolation box. The SIbehavior of both rats was recorded on-line from a monitor placed outsideon top of the box. The following parameters were scored by an observer(HPN) unaware of the substrain of rats: duration of time spent insniff-mg, following, crawling under and over other rats, but not passivebody contact (resting, sleeping). An increase of the Si time isconsidered an anxiolytic-like response.

Stress induced body weight loss. On three consecutive days, age matchedanimals from Japanese (JAP), United States (USA) and German (GER)breeders were individually transported to a novel room and remainedthere for h. On the first day a novel cage containing sawdust was usedand animals placed in a standard animal rack. On the second dayprocedure was the same except that the cage was without sawdust. Thestress procedure on the third was the same as on day 2 except that thecage was placed on the bottom of the novel room.

Statistical analysis. Data from repeated observations were analyzed bytwo-way analyses of variance for repeated measures (ANOVA) (factors:“substrain” and “change of body weight after stress” as repeatedmeasure). Data obtained from simple measures such as DPIV activity or Sitime were analyzed by one-way (factor: “substrain”) ANOVA. Asterisksindicate significant post hoc effects vs. F344USA substrain (Control)obtained by Fisher's PLSD. All data are presented as means±S.E.M.

DPIV activity in F344 substrains. Corresponding to the literature,F344GER and F344JAP substrains lack endogenous DPIV activity (FIG. 1).Thus, these rats provide a genetic model for the investigation of thephysiological role of DPIV activity in behavioral regulation.

Anxiety in the social interaction test: Those F344 substrains that lackDPIV activity (F344JAP and F344GER) spent significantly more time inactive social interaction with a novel arena (FIG. 2). Thus, the lack ofendogenous DPW-like activity mediates anxiolytic-like effects.

Stress-induced body-weight loss: F344JAP and F344GER lose significantlyless body weight after repeated 1 h transport stress. Thus, the lack ofendogenous DPIV-like activity in these substrains reduces thephysiological changes induced by moderate stress (FIG. 3).

Together, these data demonstrate that in the genetic model of the DPIVdeficient F344 rats the lack of endogenous DPIV activity causeanxiolytic-like and stress-protective effects. The pharmacologicalsimulation of DPIV deficiency, i.e. DPIV inhibitor treatment, maytherefore cause anxiolytic-like effects (see example 2).

Example 2

In the previous example we have demonstrated that anxiety andstress-responsiveness were reduced in a genetic model of DPIVdeficiency. In the present example, we report that centraladministration (i.c.v.) of the DPIV selective inhibitor P32/98 leads toanxiolytic-like effects in a well-established test of anxiety inrodents, the elevated plus maze test. We further report that also theemotionality of rats in response to novelty as measured by the openfield paradigm (Denenberg et al., 1968) is less pronounced in P32/98treated rats without affecting activity.

Animals. Male WistarF/Han (WF) rats (Central Animal Laboratory, HannoverMedical School, Hannover, Germany, weighing 350-390 g, were housed in asound-proof, temperature controlled (24.0±0.5° C.) room under specificpathogen free conditions with a 12/12 h dark/light cycle (lights on at07.00 with illumination level of 80 Lux). Food (Altromin lab chowpellets) and tap water were available ad libitum. Underketamine/xylasine (100/5 mg/kg, i.p.) anesthesia, the rats were fixed ina Kopf stereotaxic frame and implanted with cannulae (Plastic One, Inc.,Roanoke, Va., USA) above the lateral ventricle. All research and animalcare procedures were approved by the Lower Saxony district government(Hannover, Germany) and followed principles described in the EuropeanCommunity's Council Directive of 24 Nov. 1986 (86/609/EEC).

Surgery and i.c.v. application. For surgery, rats were anesthetized andprepared with i.c.v. cannulaes (coordinates: A:−0.7 mm caudal, L:1.4 mmlateral to bregma; and V:3.2 mm ventral to the skull surface; tooth bar+3.0 above ear bars) using standard stereotaxic procedures as describedin detail elsewhere (von Hörsten et al., 1998a,b,c). After a 7-dayrecovery period, successful ventricle cannulation was confirmed by anangiotensin drinking response (von Hörsten et al., 1998a). Rats showinga positive drinking response (n=40) were then habituated to experimentalhandling by daily sham injections for seven days.

Animals were randomly divided into four experimental groups(n=8-10/group), which completed different behavioral tests ofexperimental anxiety. Animals in each group were treated in an identicalway in each phase, receiving i.c.v. injections-60 mins before behavioraltesting: Artificial cerebrospinal fluid (aCSF) (Control), P32/98 (0.05nmol), P32/98 (5 nmol) and P32/98 (500 nmol). P32/98 was adjusted to thefinal concentration using buffered aCSF and applied in a volume of 5μl/min into the right lateral ventricle. The cannula was attached to aHamilton microsyringe with approximately 30 cm of polyethylene tubingand all compounds were infused at a rate of 5.0 μl/min using a TSEmultichannel infusion pump (Bad Homburg, Germany).

Response to novelty (Open field test). Differences in the response tonovelty induced by central DPIV antagonism were studied using an openfield (OF) test. The general procedure has been described elsewhere indetail (von Hörsten et al., 1993, 1998d). However, the followingmodifications were applied: During the dark phase, rats were placed in asquared 50×50 cm OF within a sound isolated box illuminated by red photolight. Spontaneous activity during a single continuous test session of15 min was recorded using a video path analyzer system (E61-21 VideoPath Analyser system, Coulbourn instruments, PA, U.S.A.). The analyzersystem determines behavior at 15 one min intervals, analyzing 14 dataelements: Wall-, corner-, quadrant 1-4-, locomotion-, rest-, andrearing-time (all in sec.), stereo-, rearing-, rotation clockwise- andcounterclockwise-events (all integers), and distance traveled (cm).Furthermore, the total incidence of fecal boli was counted after eachsession and the incidence and duration (sec) of grooming behavior wassimultaneously recorded from a video monitor by a person blind to thetreatment of the animals (von Hörsten et al., 1998c).

Elevated plus maze (EPM) testing. The elevated plus maze apparatus andthe test procedure were adapted according to Fernandes and File 1996based on general considerations and validation for use with rats. The E+apparatus (TSE Systems, Bad Homburg, Germany) was made of gray plasticand had two open arms (50×10 cm) and two enclosed arms of the same sizewith walls 40 cm high, elevated 50 cm above the ground. The maze wasequipped with light beam sensors that enabled computerized measurementof E+ performance. The maze was lit with red light bulb (Philips PF712E;1.5 Lux) placed 30 cm above maze in a way that closed arms remained inthe shadow. At start of experiment the rat was placed on centralplatform (10×10 cm), with its head facing the closed arm, and allowed tofreely explore the maze for 5 min. The following parameters werecalculated: Total numbers of arm entries (TA); entries to closed arms(CA); entries to open arms (OA); percentage frequency of entries to openarms (% OA: OA×100/TA); total trial duration (TT): 300s; duration ofstay in closed arms (closed time; CLT); percentage share of CLT in totalarms-stay duration (CLT×100/AT); duration of stay in open arms (opentime; OT) and percentage share of OT in total arm-stay duration (% OT:OT×100/AT). In addition to the standard spatiotemporal measurements,“time spent and percentage of time on the central square” were recorded(center time, CT: duration of stay on platform in seconds; percentageshare of CT in trial duration, % CT: CT×100/TT). An increase of the timespent on the open arms is interpreted as an anxiolytic response, adecrease of this parameter an anxiogenic response, whereas the number ofentries into closed arms provides an indication of general activity(Pellow et al., 1985).

Statistical analysis. For statistical analysis, behavioral raw data fromevery test minute were analyzed by two-way analyses of variance (ANOVA)for repeated measures (factors: treatment and time as a repeatedmeasurement). Data obtained as totals from a session were analyzed byone-way (factor: treatment) ANOVA. Significant post hoc effects vs. aCSF(Control) obtained by Fisher's PLSD are indicated by an asterisk. Alldata are presented as means±S.E.M.

Response to P32/98 in the open field: P32/98 had no effect on activityin the open field in a wide range of dosages (FIG. 4). As oncharacteristic of reduced emotionality in the open field, there was adose-dependent reduction of the time spent close to the wall induced byP32/98 (FIG. 5). Very low dosage of 50 pmol was already effective.

Response to P32198 in the EPM: P32/98 at low dose (50 pmol) increasedthe percentage of time spent on the open arms of the maze beingindicative for an anxiolytic-like effect (FIG. 6). Together, these dataindicate for the first time that the specific pharmacological inhibitionof CNS DPIV systems produces dose dependent anxiolytic-like effects intwo animal models of anxiety.

Example 3

In the previous examples we have demonstrated that anxiety andstress-responsiveness were reduced in a genetic model of DPIVdeficiency. We further have shown that pharmacological inhibition of CNSDPIV systems results in dose-dependent anxiolytic-like effects in theplus maze and open field paradigms of experimental anxiety. In thepresent example, we report that central administration (i.c.v.) of theDPIV selective inhibitor P32/98 itself has also dose-dependentanxiolytic-like effects in the social interaction test of anxiety. Wealso report that the magnitude of anxiolysis by P32/98 is similar tothat produced by NPY. In addition, we show that combined application ofP32/98 and NPY has additive anxiolytic-like effects in the Si test.Finally, we show that 1 h and 12 h food intake is less affected byP32/98 treatment alone and in combinations.

Animals. Male WistarF/Han (WF) rats (Central Animal Laboratory, HannoverMedical School, Hannover, Germany, weighing 330-370 g, were housed in asound-proof, temperature controlled (24.0±0.5° C.) room under specificpathogen free conditions with a 12/12 h dark/light cycle (lights on at07.00 with illumination level of 80 Lux). Food (Altromin lab chowpellets) and tap water were available ad libitum. Underketamine/xylasine (100/5 mg/kg, i.p.) anesthesia, the rats were fixed ina Kopf stereotaxic frame and implanted with cannulae (Plastic One, Inc.,Roanoke, Va., USA) above the lateral ventricle. All research and animalcare procedures were approved by the Lower Saxony district government(Hannover, Germany) and followed principles described in the EuropeanCommunity's Council Directive of 24 Nov. 1986 (86/609/EEC).

Surgery and i.c.v. application. For surgery, rats were anesthetized andprepared with i.c.v. cannulae (coordinates: A:−0.7 mm caudal, L:1.4 mmlateral to bregma; and V3.2 mm ventral to the skull surface; tooth bar+3.0 above ear bars) using standard stereotaxic procedures as describedin detail elsewhere (von Hörsten et al., 1998a, b, c). After a 7-dayrecovery period, successful ventricle cannulation was confirmed by anangiotensin drinking response (von Hörsten et al., 1998a). Rats showinga positive drinking response (n=59) were then habituated to experimentalhandling by daily sham injections for seven days.

Animals were randomly divided into two sets of four experimental groups(n=5-6/group), which completed different consecutive SI test of anxiety.Animals in each group were treated in an identical way in each phase,receiving i.c.v. injections −60 mins and −45 min before behavioraltesting with different dosages (P32/98: 5 pmol-500 nmol; NPY: 50pmol-1.6 nmol). P32/98 was adjusted to the final concentration usingbuffered aCSF and applied in a volume of 5 μl/min into the right lateralventricle. The cannula was attached to a Hamilton microsyringe withapproximately 30 cm of polyethylene tubing and all compounds wereinfused in a total volume of 5.0 μl at a rate of 5.0 μl/min using a TSEmultichannel infusion pump (Bad Homburg, Germany). The time spent inactive social interaction (SI-time), 1 h and 12 h food-intake weremeasured. Animals were repeatedly tested with randomly chosen treatmentand always-new interaction partners. Tests were separated with at least4 days from each other and always conducted in the dark cycle. Fivetests series were performed. Each test, spanning four groups (aCSF+aCSF;aCSF+NPY; P32/98+aCSF; P32/98+NPY) of 5-6 animals per condition.

Social interaction (SI) test. The SI test was carried out as firstdescribed by File (1980) and has initially been validated in thelaboratory (Kask, Nguyen, Pabst, von Hörsten, submitted). Two rats,matched for genotype and body weight, were removed from home cages andexposed to the testing environment. The arena was a squared open field(50×50×50 cm) made of aluminum, placed inside a sound isolation box(Coulbourn Instruments, Lehigh Valley, Pa.). For details of theapparatus see our previous study (von Hörsten et al., 1998c). The openfield was lit by a red photo light bulb (Philips PF712E; 1.3 Lux). Ratswere unfamiliar with the apparatus. Behavior was monitored using a videocamera placed above the field inside the testing/isolation box. The Sibehavior of both rats was recorded on-line from a monitor placed outsideon top of the box. The following parameters were scored by an observer(HPN) unaware of the substrain of rats: duration of time spent insniffing, following, crawling under and over other rats, but not passivebody contact (resting, sleeping). An increase of the Si time isconsidered an anxiolytic-like response.

Statistical analysis. Data from repeated observations (food intake) wereanalyzed by two-way analyses of variance for repeated measures (ANOVA)(factors: “substrain” and “food intake” as repeated measure). Dataobtained from simple measures such as DPIV activity or SI time wereanalyzed by one-way (factor: “substrain”) ANOVA. Asterisks indicatesignificant post hoc effects vs. aCSF+aCSF (Control) obtained byFisher's PLSD. All data are presented as means±S.E.M.

Dose dependent anxiolysis in the SI test by P32/98. Centraladministration of the DPIV selective inhibitor P32/98 (group:aCSF+P32/98: 5 pmol-500 nmol) produced “bell-shaped” dose-dependentanxiolytic-like effects in the social interaction test of anxiety (FIG.7). This demonstrates that P32/98 acts also in the SI test as a potentanxiolytic-like compound—similar to the EPM and the open field tests(see example 2). I.c.v. application of NPY (0.05-1.6 nmol) had a similaranxiolytic-like effect (FIG. 7). This finding replicates the knownanxiolytic-like action of NPY, as described in the background art. Thecomparison of P32/98-mediated effects with those of NPY indicates thatthe inhibitor is of similar potency. Interestingly, pretreatment ofP32/98 followed by NPY produced an additive effect over a wide range ofdosages (FIG. 7), suggesting that these compounds act through the samemechanism. As described in the prior art, this mechanisms is mostprobably the activation of CNS Y1 receptors.

Minor effects of P32/98 on feeding. FIG. 8 and FIG. 9 demonstrate thatat 1 h NPY produced an “u-shaped” dose-dependent feeding effect (FIG.8). P32/98 produced a mild feeding effect that only at 0.05 nmol dosereach significance (FIG. 8). Combined treatment of P32/98 followed byNPY differed not from NPY alone (except at highest non-physiologicaldosages), suggesting that the feeding effect of NPY is not mediatedthrough a mechanisms that is affected by P32/98. Most data in the priorart indicate that the dark cycle feeding effect of NPY is primarily Y5receptor mediated. Over-night food-intake was not affected by anytreatment, except at extremely high dosages (FIG. 9). Thus, centralapplication of the selective DPIV inhibitor P32/98 does not affect majorfeeding regulatory systems (i.e. the NPY Y5 receptor).

Example 4

In the previous examples we have demonstrated that anxiety andstress-responsiveness were reduced in a genetic model of DPIVdeficiency. We further have shown that pharmacological inhibition of CNSDPIV systems results in dose-dependent anxiolytic-like effects in theplus maze, open field and social interaction paradigms of experimentalanxiety in rodents. We have shown also, that the magnitude of anxiolysisby P32/98 is similar to that produced by NPY and found that combinedapplication of P32/98 and NPY has additive anxiolytic-like effects in SItest of anxiety, suggesting a potentiation of a NPY Y1 receptor mediatedeffect. In the present example we report on the mechanism for thepotentiation of NPY mediated anxiolytic-like effects at moderate dosages(P32/98, 50 nmol; NPY, 0.8 nmol) as shown in FIG. 7 of Example 3. Weconfirm that pre-treatment using the NPY Y1 receptor antagonist BIBP3226can block the P32/98-induced potentiation of the NPY mediatedanxiolytic-like effect on Si behavior and furthermore show that 1 hfeeding effects are only partly affected by blockade of the Y1 R.

Animals. Male WistarF/Han (WF) rats (Central Animal Laboratory, HannoverMedical School, Hannover, Germany, weighing 330±31 g±SD, were housed ina sound-proof, temperature controlled (24.0±0.5° C.) room under specificpathogen free conditions with a 12/12 h dark/light cycle (lights on at07.00 with illumination level of 80 Lux). Food (Altromin lab chowpellets) and tap water were available ad libitum. Underketamine/xylasine (100/5 mg/kg, i.p.) anesthesia, the rats were fixed ina Kopf stereotaxic frame and implanted with cannulae (Plastic One, Inc.,Roanoke, Va., USA) above the lateral ventricle. All research and animalcare procedures were approved by the Lower Saxony district government(Hannover, Germany) and followed principles described in the EuropeanCommunity's Council Directive of 24 Nov. 1986 (86/609/EEC).

Surgery and i.c.v. application. For surgery, rats were anesthetized andprepared with i.c.v. cannulae (coordinates: A:−0.7 mm caudal, L:1.4 mmlateral to bregma; and V3.2 mm ventral to the skull surface; tooth bar+3.0 above ear bars) using standard stereotaxic procedures as describedin detail elsewhere (von Hörsten et al., 1998a, b, c). After a 7-dayrecovery period, successful ventricle cannulation was confirmed by anangiotensin drinking response (von Hörsten et al., 1998a). Rats showinga positive drinking response (n=56) were then habituated to experimentalhandling by daily sham injections for seven days.

Animals were randomly divided into eight experimental groups(n=6-8/group), which completed one SI test of anxiety: (1)aCSF+aCSF+aCSF; (2) BIBP+aCSF+aCSF; (3) aCSF+P32/98+aCSF; (4)BIBP+P32/98+aCSF; (5) aCSF+aCSF+NPY; (6) BIBP+aCSF+NPY; (7)aCSF+P32/89+NPY; (8) BIBP+P32/98+NPY. Animals in each group were treatedin an identical way in each phase, receiving i.c.v. injections −60 minsand −55 min before behavioral testing. Experiments spanned two nightswith groups counterbalances on both dark cycles. The injection cannulawas attached to a Hamilton microsyringe with approximately 30 cm ofpolyethylene tubing and all compounds were infused in a total volume of5.0 μl at a rate of 5.0 μl/min using a TSE multichannel infusion pump(Bad Homburg, Germany). The time spent in active social interaction(SI-time), 1 h, and 12 h food-intake were measured.

Peptides and Antagonist. Rat neuropeptide Y₁₋₃₆ was obtained fromPolypeptide Laboratories (Wolfenbuttel, Germany). The NPY Y1 receptorantagonist BIBP3226 was purchased from American Peptide Company,Sunnyvale, Calif., USA (Cat#:60-1-22B). All drugs were dissolved insterile water and final dilutions were made with aCSF.

Social interaction (SI) test. The S1 test was carried out as firstdescribed by File (1980) and has initially been validated in thelaboratory (Kask, Nguyen, Pabst, von Hörsten, submitted). Two rats,matched for treatment, were removed from home cages and exposed to thetesting environment. The arena was a squared open field (50×50×50 cm)made of aluminum, placed inside a sound isolation box (CoulbournInstruments, Lehigh Valley, Pa.). For details of the apparatus see ourprevious study (von Hörsten et al., 1998c). The open field was lit by ared photo light bulb (Philips PF712E; 1.3 Lux). Rats were unfamiliarwith the apparatus. Behavior was monitored using a video camera placedabove the field inside the testing/isolation box. The SI behavior ofboth rats was recorded on-line from a monitor placed outside on top ofthe box. The following parameters were scored by an observer (HPN)unaware of the substrain of rats: duration of time spent in sniffing,following, crawling under and over other rats, but not passive bodycontact (resting, sleeping). An increase of the SI time is considered ananxiolytic-like response.

Statistical analysis. Data obtained from the measures SI time and 1 hfood intake were analyzed by one-way (factor: “treatment”) ANOVA. Inaddition, three factorial (factors: “Antagonist”, “Inhibitor”, “NPY”)ANOVA was performed to confirm general conclusions (data not shown).Asterisks indicate significant post hoc effects vs. aCSF+aCSF+aCSF(Control) while “#” signs indicate a significant difference of Y1 Rantagonist treatment vs. the corresponding treatment without antagonistobtained by Fisher's PLSD. The level of significance in post hoccomparisons is indicated by asterisks with “*”=p<0.05; “**”=p<0.01;“***”=p<0.001) and by “#” symbols with “#=p<0.05; “##”=p<0.01;“###”=p<0.001). All data are presented as means±S.E.M.

NPY Y1 receptor mediated potentiation of NPY-induced anxiolysis in theSI test by P32/98 pre-treatment. Combined central administration ofP32/98 at a dose of 50 nmol and NPY at a dose of 0.8 nmol dramaticallypotentiated the anxiolytic-like effect of NPY in the SI test (FIG. 10).This finding replicates at corresponding dosages the observation shownin FIG. 7. At this dose p32/98 treatment alone acts not anxiolytic-like.However, the endogenous social investigatory behavior, theanxiolytic-like effect of NPY and the potentiated anxiolysis induced bycombined P32/98+NPY treatment were all antagonized by Y1 receptorblockade. This indicates a tonic regulation of anxiety levels via theNPY Y1 receptor in the CNS and furthermore proofs that theanxiolytic-like effect of NPY as well as the potentiated anxiolysisinduced by combined treatment all are primarily Y1 receptor mediated.Spontaneous feeding and NPY-induced feeding are only partly mediated bythe Y1 R and the non-significant slight increase of feeding induced byP32/98 treatment is blocked by Y1 R antagonisms (FIG. 11).

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1. A method of treating schizophrenia or depression, the methodcomprising administering to a subject in need thereof, a therapeuticallyeffective amount of a composition comprising an inhibitor of dipeptidylpeptidase IV (DP IV), or a pharmaceutically acceptable salt thereof. 2.A method in accordance with claim 1, wherein administering comprisesadministering orally.
 3. A method in accordance with claim 7, whereinadministering comprises administering parenterally.
 4. A method inaccordance with claim 3, wherein administering parenterally comprisesadministering intravenously.
 5. A method in accordance with claim 3,wherein administering parenterally comprises administering by injection.6. A method in accordance with claim 3, wherein administeringparenterally comprises administering by infusion.
 7. A method inaccordance with claim 3, wherein administering parenterally comprisesadministering by inhalation.
 8. A method in accordance with claim 1,wherein administering comprises administering by suppository.
 9. Amethod in accordance with claim 1, wherein the inhibitor is selectedfrom the group consisting of N-(N′-substitutedglycyl)-2-cyanopyrrolidines, L-threo-isoleucyl thiazolidine,L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine andL-allo-isoleucyl pyrrolidine.
 10. A method in accordance with claim 1,wherein the inhibitor is present in a physiologically compatible drugdelivery vehicle.
 11. A method in accordance with claim 1, wherein themethod further comprises administering neuropeptide Y to the patient.12. A method in accordance with claim 1, wherein the compositioncomprises a prodrug form of the DP IV inhibitor.
 13. A method inaccordance with claim 11, wherein the neuropeptide Y and the inhibitorof dipeptidyl peptidase IV (DP IV) are administered simultaneously. 14.A method in accordance with claim 13, wherein the composition furthercomprises neuropeptide Y.
 15. A method in accordance with claim 11,wherein the neuropeptide Y and the inhibitor of dipeptidyl peptidase IV(DP IV) are administered separately.
 16. A method in accordance withclaim 1 wherein the composition further comprises at least onepharmaceutically acceptable carrier or excipient.
 17. A method inaccordance with claim 1, for the treatment of schizophrenia.
 18. Amethod in accordance with claim 1, for the treatment of depression. 19.A method in accordance with claim 1, for the treatment of schizophreniaand depression.